Trypanosoma brucei brucei causes Nagana in cattle but is non-infectious to humans because of its susceptibility to the cytotoxic activity of normal human serum. This innate immune activity is due to a minor subclass of high-density lipoprotein (HDL) termed the Trypanosome Lytic Factor (TLF). TLF contains apolipoprotein A-1 (apoA-1), a protein found in all HDLs, and two primate specific proteins, haptoglobin related protein (Hpr) and apolipoprotein L-1 (apoL-1). Both Hpr and apoL-1 have been shown to be necessary for maximal trypanosome killing in vitro and in a transgenic mouse model. Hpr is a hemoglobin (Hb) binding protein and Hb is an essential co-factor for trypanosome killing by TLF. The cellular pathway of TLF mediated lysis of T. b. brucei initiates with binding of TLF to a high affinity receptor (Tb927.6.440) located in the flagellar pocket. Following binding TLF is rapidly taken up and localized to the lysosome. Within the acidified lysosome TLF is activated and kills T. b. brucei by destabilization of the lysosomal membrane. The human sleeping sickness parasite Trypanosoma brucei rhodesiense has evolved a defense against TLF. T. b. rhodesiense produces a potent inhibitor of TLF, Serum Resistance Associated protein (SRA), which binds apoL-1 and neutralizes the activity of TLF. Trypanosoma brucei gambiense also infects humans but lacks SRA and, as shown in this proposal, resists TLF killing by down regulation of the TLF receptor. In this proposal, we outline a number of experiments that will provide important information on the mechanism of assembly of TLF and how its composition might be altered to produce variant TLFs with activity against the human sleeping sickness parasites (Aim 1). To better understand the biophysical basis for membrane destabilization by native TLF and its constituent proteins we will use model liposomes of defined composition in fluorescence based assays to measure TLF induced changes in lipid dynamics (Aim 2). Finally, we will investigate the cellular, molecular and biochemical basis of SRA-dependent and SRA-independent resistance to TLF. In addition to resolving a longstanding question concerning how SRA inhibits TLF activity we will investigate the mechanism of T. b. gambiense resistance to TLF (Aim 3). Our long-term goals are to develop a better understanding of the mechanism of TLF killing and how the human sleeping sickness parasites evade this activity. This may allow us to develop novel approaches for the treatment of this important human disease.